Coordinated DNA 5-mC and RNA m5C methylation epigenetically regulates MZF1 splice variants to drive EGFR-TKI resistance.

Acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) severely limits clinical efficacy. Myeloid zinc finger 1 (MZF1) inhibited EGFR phosphorylation and internalization by maintaining zinc homeostasis, which suggested its potential as a therapeutic target for resensitization. The two MZF1 splice variants exhibited functional divergence. MZF1S demonstrated significantly reduced Zn2+-binding capacity compared with MZF1L owing to C2H2 domain deletion. Although MZF1S was specifically upregulated in resistant patients, MZF1L predominated in treatment-sensitive cohorts, the mechanisms regulating their differential expression remained uncharacterized. We identified NSUN7/YBX1-mediated RNA m5C methylation as a critical driver of resistance. This modification promoted competitive binding of SRSF1 over SRSF3 to MZF1 pre-mRNA and enhanced MZF1S production. Overlapping loci co-regulated by RNA m5C and DNA 5-methylcytosine methylation in the MZF1 5'-untranslated region suggested coordinated epigenetic control of alternative splicing. Characterization of these loci revealed a UHRF1/DNMT1-NSUN7/YBX1 axis that synergistically regulated splice variant selection. Targeting this axis suppressed MZF1S while restoring MZF1L expression, which resensitized resistant cells to EGFR-TKIs. Our findings established a dual-layer epigenetic mechanism governing alternative splicing in drug resistance and proposed methylation-regulated splice variants as biomarkers and therapeutic targets for overcoming EGFR-TKI resistance in non-small-cell lung cancer.